Over the years well-established methods have been developed. For instance, the separation of serum proteins in a series of characteristic bands, commonly referred to as gamma, beta, alpha-1 and 2 and albumin by electrophoresis is well know to those skilled in the art and is well described in a wide range of reference materials, such as the handbook ‘Protein Electrophoresis in Clinical Diagnosis’ by David F. Keren, 2003, Arnold Publications.
Commonly known methods include the use of techniques wherein a specific compound is subjected to a complexation with a specific binding partner, such as an antibody against that compound. Such techniques may be used to remove the compound from the sample prior to electrophoretic separation (typically referred to as immunosubtraction when an antibody is used as a binding partner) or to modify the effective electrophoretic mobility of that compound during electrophoresis (referred to as immunodisplacement when an antibody is used as a binding partner).
The target compound that forms a complex with the binding partner may be a compound that is to be identified, e.g. a monoclonal immunoglobulin which may be an indicator for a monoclonal gammopathy. In cases where the binding partner is an antibody this method may be referred to as immunotyping.
Alternatively, the target compound may be an interfering factor, i.e. potentially disturbing the analysis of a sample for another compound of which the presence is to be determined, by, for example, co-migrating with the analyte of interest or in other ways masking it's presence, e.g. a gamma-globulin which may migrate very close to carbohydrate-deficient isoforms of transferrin and hence impede their use as a diagnostic marker for excessive alcohol consumption
Capillary electrophoresis is a specific form of electrophoresis, wherein a capillary is used to perform the electrophoresis. Capillary electrophoresis offers advantages such as short analysis times and a high resolution. A method for the capillary electrophoretic analysis of a sample comprising a compound of which the presence is to be determined (i.e. analyte), wherein use is made of an antibody as a binding partner for the analyte is described in WO 95/20160. The method involves:
(a) separating a first portion of the sample into constituent analyte parts by capillary electrophoresis, and detecting said parts;
(b) admixing a second portion of said sample with at least one specific binding partner to a predetermined candidate analyte, said specific binding partner having an electrophoretic mobility different from that of said candidate analyte, hence conferring to the resulting complexes formed an electropheric mobility different to the unbound analyte;(c) separating said second portion into constituent parts by capillary electrophoresis, and detecting said parts; and,(d) comparing the separated constituent parts of step (c) with the separated constituent parts of step (a).
As a specific binding partner preferably an antibody is used that has been chemically modified with an anhydride, such as succinic anhydride, to provide the antibody with additional carboxylic acid functions, negative at alkaline pH. Under the analytical conditions described in the example (pH 10) the overall negative charge of the modified antibody is therefore increased, compared to the unmodified antibody. However, as shown in FIG. 1 of WO95/20160, the modified antibody still migrates closely to human immunoglobin (IgG) and in particular the complex of the modified antibody with IgG is not fully separated from uncomplexed IgG. It is apparent that in a real biological sample which is to be analysed for the presence of one or more serum proteins (e.g. blood serum, urine, cerebrospinal fluid), the electrophoretic migration of the modified antibody and in particular of the complex of antibody and immunoglobin in the sample would be such that they may co-migrate with serum proteins, for instance, another immunoglobin, a transferrin, albumin or bis-albumin, for which the sample is to be analysed.
US 2005/0164302 A1 proposes an alternative method of separating the constituents of a biological sample and carrying out immunodisplacement to allow typing of monoclonal proteins which may be present in the analysed biological sample. It is mentioned that the method allows displacement outside the zone corresponding to the migration profile for the proteins of the sample, in particular outside the globulin migration zone. This is said to be accomplished by modifying the binding partner (an antibody) in a specific way. The specific modifications shown are a modification of the antibodies with tricarboxylic anhydride and modification with mellitic acid. From the Examples it is apparent that the modified antibody has an effect in that immunodisplacement takes place, but it is also apparent that modified antibody or complex of modified antibody and immunoglobulin of the sample is not fully separated from other proteins in the sample. Notably, several electropherograms, e.g. FIGS. 2e, 4b, 5a, 9a and 9b of US 2005/0164302 A1, suggest an overlap with albumin and possibly other proteins migrating between albumin and the immunoglobulins of the sample. Further, in a test performed by the present inventors the migration time of an unmodified antibody (IgG) and an antibody (IgG) modified with benzene tricarboxylic acid anhydride in a buffer at pH 10 (comprising 3-cyclohexylamino-1-propanesulphonic acid (CAPS) and N-Tris(hydroxymethyl)methyl-3-aminopropanesulphonic acid (TAPS)), it was found that the modified antibody had a migration time (peak top time) that was similar to the migration time of albumin, indicating that the modified antibody would not be baseline-separated from albumin in a blood serum sample. Further, this is an indication that a complex of the modified antibody and an immunoglobin in a serum sample may have a migration time in between the migration time of immunoglobins and the migration time of albumin. Thus, the complex would likely at least partially co-migrate with other proteins, e.g. the alpha-band serum proteins, in an electrophoretic analysis using such a buffer. Thus, it remains a challenge to avoid undesired co-migration of the complex, especially in the separation of a complicated sample, such as a blood serum or another biological sample.
The present disclosure is directed toward overcoming one or more of the problems discussed above.